Db wax 126 7012

The degradation of casein fractions was evaluated by capillary zone electrophoresis (CZE) using a P/ACE^TM MDQplus equipment (AB Sciex, Milan, Italy) and a hydrophilically coated capillary column DB-WAX126-7012 (50 cm length, 50 µm i.d., 0.05 µm coating, 100 × 800 µm slit opening) (Agilent Technologies, Milan, Italy). The analytical conditions were those described by D’Incecco et al. [14 (link)]. Briefly, 1 g of grated cheese was added with 10 mL of sample buffer, kept at room temperature for 4 h, then diluted 1:5 with the same buffer and filtered with a 0.22 µm PVDF membrane filter (Merck, Milan, Italy) immediately before analysis. Separation was carried out with the column temperature set at 45 °C and using a linear gradient from 0 to 30 kV for 4 min and a subsequent step at a constant voltage of 30 kV. The electropherograms were recorded at 214 nm and the normalized peak areas (peak area counts/migration time) were considered. Peak area ratios were calculated between selected casein breakdown fragments and the corresponding parent casein fractions. In particular, the ratios αs1-I-CN/αs1-CN and Σγ-CN/Σβ-CN described by D’Incecco et al. [14 (link)] were considered as indicators of the primary proteolysis of αs1- and β-casein fractions, respectively.

All samples were dispersed (600 mg/10 mL) in 10M Urea/DTT buffer (pH 8.6) and kept at room temperature for 4 h. Solubilized samples were filtered through a 0.2 µm membrane filter (Millipore, Milan, Italy) before analysis. The CZE conditions previously described [46 (link)] were followed using a Beckman P/ACE System MDQplus, equipped with a 50 cm fused silica column (DB-WAX 126-7012, Agilent Technologies, Milan, Italy). The anode pressure injection was 0.5 psi for 20 s. Samples were run at 45 °C using a 4-min linear gradient from 0 to 30 KV, then the current voltage was kept constant at 30 KV for 56 min. Detection was carried out at 214 nm and the correct peak areas were calculated as peak area/migration time. Samples were analyzed in duplicates.

The extent of proteolysis in the DC and NDC was investigated through capillary zone electrophoresis (CZE), which allows the separation of intact casein fractions as well as major peptides. The conditions described in [28 (link)] were adopted. Briefly, 1 g of grated cheese was dissolved in 10 mL of urea-DTT buffer (pH 8.6) at room temperature for 4 h. The solubilised samples were further diluted 1:5 with the same buffer and then filtered using a 0.22 µm membrane filter (Millipore, Burlington, MA, USA) before analysis. The separation was carried out at 45 °C using a Beckman P/ACE System MDQplus equipped with a 50 cm fused silica column (DB-WAX 126-7012, Agilent Technologies, Milan, Italy). The detection was carried out at 214 nm, and the corrected peak areas, calculated as the peak area/migration time, were used to calculate the ratios between the selected peptides and their parent casein fractions [28 (link)].